The Peptide Semax Affect The Expression Genes Related To The Immune and Vascular System

Medvedeva et al.
BMC Genomics 2014, 15:228

http://www.biomedcentral.com/1471-2164/15/228
RESEARCH ARTICLE Open Access
The peptide semax affects the expression of

genes related to the immune and vascular
systems in rat brain focal ischemia: genome-wide
transcriptional analysis
Ekaterina V Medvedeva1*, Veronika G Dmitrieva1, Oksana V Povarova2,3, Svetlana A Limborska1,2, Veronika I Skvortsova2,
Nikolay F Myasoedov1 and Lyudmila V Dergunova1,2
Abstract
Background: The nootropic neuroprotective peptide Semax (Met-Glu-His-Phe-Pro-Gly-Pro) has proved efficient in
the therapy of brain stroke; however, the molecular mechanisms underlying its action remain obscure. Our
genome-wide study was designed to investigate the response of the transcriptome of ischemized rat brain cortex
tissues to the action of Semax in vivo.
Results: The gene-expression alteration caused by the action of the peptide Semax was compared with the gene
expression of the “ischemia” group animals at 3 and 24 h after permanent middle cerebral artery occlusion
(pMCAO). The peptide predominantly enhanced the expression of genes related to the immune system. Three
hours after pMCAO, Semax influenced the expression of some genes that affect the activity of immune cells, and,
24 h after pMCAO, the action of Semax on the immune response increased considerably. The genes implicated in
this response represented over 50% of the total number of genes that exhibited Semax-induced altered expression.
Among the immune-response genes, the expression of which was modulated by Semax, genes that encode
immunoglobulins and chemokines formed the most notable groups.
In response to Semax administration, 24 genes related to the vascular system exhibited altered expression 3 h after
pMCAO, whereas 12 genes were changed 24 h after pMCAO. These genes are associated with such processes as
the development and migration of endothelial tissue, the migration of smooth muscle cells, hematopoiesis, and
vasculogenesis.
Conclusions: Semax affects several biological processes involved in the function of various systems. The immune
response is the process most markedly affected by the drug. Semax altered the expression of genes that modulate
the amount and mobility of immune cells and enhanced the expression of genes that encode chemokines and
immunoglobulins. In conditions of rat brain focal ischemia, Semax influenced the expression of genes that promote
the formation and functioning of the vascular system.
The immunomodulating effect of the peptide discovered in our research and its impact on the vascular system
during ischemia are likely to be the key mechanisms underlying the neuroprotective effects of the peptide.
Keywords: Semax, Pro-Gly-Pro, Focal cerebral ischemia, Expression Beadchip gene array, Gene expression, Immune
cells, Immunoglobulins
* Correspondence: medvedevaekaterina@yandex.ru
1
Human Molecular Genetics Department, Institute of Molecular Genetics,
Russian Academy of Sciences, Moscow, Russian Federation
Full list of author information is available at the end of the article
© 2014 Medvedeva et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the
Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly credited.
Medvedeva et al. BMC Genomics 2014, 15:228 Page 2 of 12
Background
Ischemic brain stroke is one of the major contributors to
mortality and disability worldwide. As the result of a
critical reduction of blood flow in the brain, it causes
massive loss of neurons and leads to the formation of
the necrotic core and the penumbra zone [1].
One of the drugs that is effectively employed currently
in cerebral stroke therapy is the Semax (Met-Glu-His-
Phe-Pro-Gly-Pro), which is a synthetic peptide consisting
of a fragment of ACTH(4–7) and the C-terminal tripep-
tide Pro-Gly-Pro (PGP). Studies have shown that Semax
promotes the survival of neurons during hypoxia [2]
and glutamate neurotoxicity [3]. It also shows neuropro-
tective properties and contributes to mitochondrial stabil-
ity under stress induced by the deregulation of calcium
Figure 1 Genes that were up- and downregulated. The x-axis
ion flow [3]. The action of Semax causes the inhibition of shows the condition of the experiment and time after pMCAO.
nitric oxide synthesis [4], improves the trophic supply of The y-axis represents the number of genes that exhibited changed
the brain [5], and protects the nervous system effectively expression in these conditions. The cut-off of gene-expression
against diseases of the optic nerve [6]. This peptide also changes was 1.50.
possesses nootropic activity [7].
However, the molecular mechanisms underlying the Note that different gene groups exhibited Semax-induced
action of Semax remain unclear. We have previously alteration of expression at 3 h and 24 h. The overlapping
shown the effect of Semax on the expression of genes group comprised only 10 genes with responses to the
that encode neurotrophic factors and their receptors in peptide that were contradictory (Table 1).
an experimental model ischemia in the rat brain [8,9].
This genome-wide study was performed to elucidate
Molecular functions of the protein products of genes with
the transcriptome response of the ischemized focal tis-
altered expression under Semax treatment
sues of the rat brain to the action of Semax in vivo. The
The grouping of the genes according to the molecular
main task of our study was to identify genes with an al-
functions of their products and to the iReport Web tool
tered expression that accounts for the positive effect
revealed that the expression of transcription regulator
exerted by Semax in the treatment of patients with is-
genes was predominantly enhanced, and that that of genes
chemic stroke [10,11].
encoding transmembrane receptors, transport proteins,
and various enzymes was decreased 3 h after the onset of
Results
ischemia under Semax treatment (Figure 2A); about 39%
Semax-induced increase and decrease in gene expression
of the genes with altered expression encoded proteins with
The genome-wide expression changes induced by Semax
molecular functions that were unrelated to the groups
in rat brain cortex tissues damaged by focal ischemia
presented or were not yet identified. Gene expression was
were studied using the genome-wide RatRef-12 Expres-
increased mostly at 24 h (Figure 2B). The largest increase
sion BeadChip (Illumina, USA), which contains 22,226
in expression was observed for immunoglobulin and cyto-
genes, according to NCBI. Data on the gene expression
kine (chiefly chemokine) genes (Table 2). The molecular
changes induced by the peptide were compared with the
functions of 24% of the protein products of the genes that
gene expression levels in the “ischemia” group at 3 and
exhibited altered expression levels were unknown.
24 h after pMCAO.
The largest number of genes (96) that exhibited al-
tered expression (cut-off 1.50) in response to Semax ad- Biological processes that were significantly associated
ministration was detected 3 h after the onset of ischemia with the genes that exhibited altered expression levels in
(Additional file 1); moreover, the amount of the genes response to the administration of the peptide
with decreased expression was insignificantly larger than We used an online program [12] to analyze genes with
that of those with increased expression (Figure 1). altered expression in response to the intermittent admin-
Semax altered the expression of 68 genes 24 h after oc- istration of Semax to ischemized animals. This led to
clusion (Additional file 2): the expression of 51 genes the identification of several biological processes that
was increased and the amount of genes with decreased were associated with the gene expression changes observed
expression was considerably lower than that observed at (Figure 3). The reliability of these processes was calculated
3 h after the onset of ischemia. by Fisher’s exact test.
Table 1 Comparison of genes that exhibited Semax-induced alteration of expression levels after pMCAO
Gene symbol Ischemia + semax 3 h Ischemia + semax 24 h ENTREZ GENE_ID
lg (Ratio) P-value lg (Ratio) P-value
RGD1562905 0.58 1.1E-04 −0.53 2.1E-34 292539
Adamts1 0.34 5.2E-11 −0.25 7.5E-09 79252
Zfp36 0.24 5.5E-03 −0.16 2.0E-04 79426
Ptprcap −0.28 2.3E-03 0.29 9.8E-08 499300
Ccdc53 −0.29 1.3E-06 0.18 4.6E-03 299707
RT1-Ba −0.29 3.1E-07 0.36 1.5E-15 309621
Cd74 −0.30 1.6E-09 0.21 5.3E-06 25599
RT1-A1 −0.30 7.6E-10 0.29 2.8E-11 24973
H2-Ea −0.31 4.1E-10 0.15 7.3E-04 294269
Igh-1a −0.79 2.6E-30 0.47 3.5E-26 299352
The table shows the genes that exhibited significantly changed expression under Semax treatment at both time points (3 and 24 h). The logarithmic transformed
value visually represents the symmetric changes in expression levels: negative value indicates decrease and positive value – increase of the gene expression level.
The cut-off of gene-expression changes was 1.40. Ratio – relative expression level.
Three hours after pMCAO, Semax exerted considerable Another remarkable group of genes with Semax-induced
influence on various general biological processes (prolifer- alteration in expression levels consisted of genes involved
ation, differentiation, and migration of cells), on vascular in the vascular system. The expression of 24 and 12 genes
system processes, brain cell processes, and on the immune was altered 3 and 24 hours after pMCAO, respectively
system (Figure 3A). Twenty-four hours after occlusion, (Table 4).
similar to observed effects 3 h after the procedure, Semax, Genes that regulate the levels of Ca2+ formed a separate
acting in conditions of focal ischemia, altered the expres- group of genes exhibiting a significant Semax-induced
sion of genes involved in cell proliferation and migration. alteration of expression 24 h after occlusion (Table 3).
One day after the occlusion, however, unlike at 3 h after
the procedure, additional processes supplemented the Discussion
general processes, namely, the organization of the cyto- Different profiles of gene expression elicited by Semax
skeleton, tissue development, and the quantity of metal administration 3 and 24 h after pMCAO
(Figure 3B). Special attention should be drawn to the pro- The dynamic state of mRNA expression in mammalian
cesses that were most significantly associated with im- tissues changes during pathophysiological processes and
mune cell activity and calcium ion regulation, namely, the after the introduction of medicinal peptides into the or-
migration and attraction of dendritic cells (DCs), the at- ganism. In this context, we studied transcriptome changes
traction of leukocytes (Figure 3B), and the regulation of caused by the action of neuropeptide Semax in the ische-
the levels of Ca2+ (Figure 3B, Table 3). mized rat brain cortex. This genome-wide study showed
Semax altered the expression of genes related to the that Semax affected the transcript level of several dozens
immune system to a large degree (Table 2). In conditions of genes 3 and 24 h after pMCAO; however, the functional
of experimental focal ischemia, the action of Semax ob- significance of many of them remains unknown.
served 3 h after the onset of ischemia influenced the ex- Three hours after pMCAO was used in the analysis as
pression of several genes that are involved in the regulation a time point inside the therapeutic window of the drug
of the activity of immune cells: macrophages, neutrophils, and within the response time of early-response genes
and lymphocytes (Figure 3A). The effect of Semax on [13]. At that time point, we found a considerable alter-
the immune response was increased significantly 24 h ation of the expression of genes encoding transcription
after pMCAO. The genes involved in this process repre- factors that could set off new signal pathways that allow
sented over 50% of the total amount of the genes that the correction of the destructive processes that devel-
exhibited altered expression levels. Semax-induced up- oped after vascular occlusion. During the active stage of
regulation of transcripts was observed for a majority of ischemia and the response of late-response genes, i.e.,
the immune-response genes; among these, immuno- 24 h after pMCAO, we observed increased levels of tran-
globulin genes formed the most prominent group, with scripts encoding transmembrane receptors and enzymes,
half of them exhibiting the highest amplitude of expres- especially cytokines and immunoglobulins. One can pre-
sion alteration among the genes for which the level of sume that processes initiated by transcription factors
transcripts was affected by the peptide (Table 2). during the first hours of therapy of ischemized animals
Figure 2 Molecular functions associated with the up- and downregulated genes. The x-axis shows the categories of molecular functions.
The y-axis represents the number of genes associated with selected cellular functions. The genes that were upregulated are indicated by dark
columns, whereas the genes that were downregulated are depicted by bright columns. The cut-off of gene-expression changes was 1.50. A. Data
obtained 3 h after pMCAO for the ischemic rat cortex under Semax treatment; B. Data obtained 24 h after pMCAO for the ischemic rat cortex
under Semax treatment.
Table 2 Genes related to the immune system and exhibited Semax-induced alteration of expression levels
Genes related to the immune system (9 genes): Gene symbol ENTREZ GENE_ID Fold change P-value
MHC (major histocompatibility class) I and II:
RT1 class I, CE15 (RT1-CE15), mRNA. RT1-CE15 414789 0.43 4.0E-06
RT1 class I, M6, gene 2 (RT1-M6-2), mRNA. RT1-M6-2 365527 0.64 3.0E-03
RT1 class II, locus Db1 (RT1-Db1), mRNA. RT1-Db1 294270 0.55 7.6E-04
RT1 class II, locus Ba, mRNA. RT1-Ba 309621 0.51 3.1E-07
3h CD74 antigen (invariant polpypeptide of MHC class II antigen-associated), mRNA. Cd74 25599 0.50 1.6E-09
RT1 class Ia, locus A1, mRNA. RT1-A1 24973 0.50 7.6E-10
histocompatibility 2, class II antigen E alpha, mRNA. H2-Ea 294269 0.49 4.1E-10
Others:
Prostaglandin-endoperoxide synthase 2, mRNA. Ptgs2/Cox2 29527 2.00 8.3E-34
Intercellular adhesion molecule 1, mRNA. Icam1 25464 1.61 9.9E-05
Genes related to the immune system (36 genes):
immunoglobulins:
Similar to immunoglobulin heavy chain variable region, mRNA. LOC500734 500734 15.37 7.5E-11
Similar to immunoglobulin kappa-chain, mRNA. LOC500172 500172 11.57 2.1E-34
Similar to Immunoglobulin kappa-chain VJ precursor, mRNA. LOC500161 500161 8.31 2.1E-34
Similar to gamma-2a immunoglobulin heavy chain, mRNA. LOC362796 362796 8.20 2.1E-34
Similar to IG kappa-chain V-V region K2 precursor, mRNA. LOC500180 500180 4.67 4.8E-08
Similar to Igh-1a_predicted protein, mRNA. LOC503073 503073 3.67 7.3E-14
Similar to NGF-binding Ig light chain, mRNA. LOC502820 502820 3.60 5.6E-23
Immunoglobulin heavy chain 1a (serum IgG2a), mRNA. Igh-1a 299352 2.97 3.5E-26
24h Similar to NGF-binding Ig light chain, mRNA. LOC500183 500183 2.91 3.8E-22
Similar to immunoglobulin light chain variable region, mRNA. LOC363828 363828 2.52 9.7E-03
Similar to IG light chain Vk region Y13-259, mRNA. LOC500181 500181 1.85 3.2E-07
Similar to Ig kappa light chain precursor, mRNA. LOC500177 500177 1.76 3.2E-06
Similar to immunoglobulin light chain variable region, mRNA. LOC502831 502831 0.34 6.3E-06
Chemokines:
Chemokine (C-X-C motif) ligand 13, mRNA. Cxcl13 498335 4.12 1.6E-08
Chemokine (C-C motif) ligand 5, mRNA. Ccl5 81780 1.98 6.5E-05
Table 2 Genes related to the immune system and exhibited Semax-induced alteration of expression levels (Continued)
MHC (major histocompatibility class) I and II:
RT1 class I, A3, mRNA. RT1-A3 309627 1.96 4.6E-06
RT1 class I, T24, gene 4, mRNA. RT1-149 414784 1.86 3.8E-10
Histocompatibility 2, M region locus 10.6, mRNA. H2-M10.6 414787 1.77 2.0E-06
CD74 antigen (invariant polpypeptide of MHC class II antigen-associated), mRNA. Cd74 25599 1.63 5.3E-06
Entrez Gene is NCBI's repository for gene-specific information. In the table, P-values are in the form of an exponential number format.
were developing further. Several similar processes were rat brain cortex. Several studies had shown previously
observed in the course of the associative analysis of bio- that intravenous immunoglobulin (IVIG) has a strong
logical processes. neuroprotective effect against ischemic impairment of
the brain [23]. It is believed that IVIG application is one
Response of immune system cells to Semax of the options for acute brain stroke therapy [24].
administration and regulation of the expression of genes Whether or not the neuroprotective effect of Semax can
encoding chemokines and immunoglobulins be a consequence of the enhancement of the expression
The detailed analysis of genes that exhibited altered of immunoglobulin should be addressed in future
levels of expression 3 and 24 h after pMCAO allowed studies.
the determination of the effect of Semax on various bio- Cytokines (particularly chemokines), which are one of
logical processes that were categorized under broad sub- the most important participants in the immune re-
groups, namely, general category, brain cell, immune, sponse, were also expressed actively 24 h after pMCAO
and vascular processes. The neuroprotective and noo- under the influence of Semax in the region of the brain
tropic properties of Semax were previously associated where the ischemic lesion was localized. Many reports
only with events that are directly relevant to nervous tis- have described chemokine expression in astrocytes, micro-
sues [2,7,11]. Here, we uncovered the action of Semax glia, and even neurons [25]. It is accepted that some
on the immune system for the first time. Three hours chemokines and their receptors are involved in various
after pMCAO, Semax acted on microglia and immune neurodegenerative diseases [26], including ischemic
system cells. The process of leukocyte activation was af- brain damage [27].
fected most significantly (P-value = 7.6 × 10−8) in the im- Recent research has shown that chemokines are a
mune response subgroup. The processes that developed unique class of neuromediators that ensure the cross-
24 h after pMCAO, which involved leukocytes, remained talk between neurons and cells from their surrounding
significant. In addition, Semax affected DCs, the pres- microenvironment [28]. In accordance with this, the
ence of which in rat cerebral hemisphere ischemia- division of chemokines into pro- and anti-inflammatory
damaged tissues had been reported by other researchers factors seems to be too simplified and gives rise to con-
[14]. DCs constitute a heterogeneous class of antigen- tradicting opinions regarding the neuroprotective and
presenting cells that are capable of immune response neurodegenerative functions of chemokines [29]. En-
initiation [15] and cytokine production [16]. hanced expression of chemokine-encoding genes is one
Both inflammation and immune response play an im- more evidence in favor of the possible existence of a
portant role in ischemic stroke. It is well known that the Semax immunomodulatory effect in conditions of focal
penetration of inflammatory/immune cells into brain tis- cerebral ischemia of the brain.
sues during the postischemia hours aggravates the situ- Semax-induced activation of chemokine genes pre-
ation [17-19]. In addition, no data have been reported to sumably accounted for the altered transcript level of
date indicating the presence of a specific cause-and- genes associated with the regulation of the quantity of
effect relationship between the penetration of leukocytes Ca2+ (Figure 3B, Table 3). The ability of some chemo-
into the damaged tissues and the pathogenesis of the is- kines to raise the level of intracellular Ca2+, which plays
chemia itself [20]. However, some studies support the a messenger role in nervous tissues, has been described
neuroprotective abilities of immune cells [21,22]. in several studies [30,31]. A study that used human neu-
It should be mentioned that the most noticeable im- trophils [32] offered experiment-based support of the ef-
mune response to Semax action was observed at 24 h fect of Semax on the Ca2+ level in cells, and showed an
after pMCAO. A high level of immunoglobulin tran- increase in Ca2+ levels caused by the effect of Semax on
scripts was found at that time point in the ischemized the mechanisms that regulate Ca2+-dependent channels.
Figure 3 Biological processes based on the genes that exhibited alteration in their expression levels under Semax treatment. The x-axis
is the absolute value of the log transformed P-value, which means that a smaller P-value has a larger positive value on the x-axis. Significance was
determined using Fisher’s exact test (P-value < 0.01). A value of 2 on the x-axis is equivalent to a P-value of 0.01. The y-axis shows the categories
of biological processes that were related to lists of genes that exhibited changed expression in the experimental conditions. A. Data obtained 3 h
after pMCAO for the ischemic rat cortex under Semax treatment; B. Data obtained 24 h after pMCAO for the ischemic rat cortex under
Semax treatment.
It is well known that ischemia-induced energy deple- contributes to neuron survivability in the conditions of
tion in cells results in disturbed operation of potential- glutamate neurotoxicity that accompany ischemia [3].
dependent calcium channels and Na+/Ca2+ pumps, exces- Some authors have suggested that cellular death is caused
sive intracellular accumulation of Ca2+ ions, and neuronal by the Ca2+ influx pathway, and not by Ca2+ load [34].
death [33]. However, it has been shown that Semax Possibly, the neuroprotective effect of Semax on ischemia-
Table 3 Genes related to the quantity of Ca2+ and exhibited Semax-induced alteration of expression levels (24 h)
Definition (12 genes) Gene symbol ENTREZ GENE_ID Fold change P-value
Transthyretin (Ttr), mRNA. Ttr 24856 20.55 2.1E-34
Thyrotropin releasing hormone, mRNA. Trh 25569 1.95 8.5E-08
Secreted phosphoprotein 1, mRNA. Spp1 25353 1.58 3.4E-06
Gastrin releasing peptide, mRNA. Grp 171101 1.53 1.3E-03
damaged nervous tissues includes the impact of Ca2+ feature of postischemic revascularization [47]. Three
penetration into the cell on the regulatory processes. This hours after pMCAO, Semax affected the expression of
idea is based on recent studies of the neuroprotective ef- genes involved in vasculogenesis and the transcription
fect of Ca2+-activated potassium channels in conditions of levels of genes associated with hematopoiesis and the
brain ischemic damage [35,36]. migration of endothelial cells. Some signal pathways are
The opinions on the role of the immune system in the well known to be active in both hematopoiesis and vas-
pathogenesis of ischemia vary. Studies are available re- culogenesis [48]. Moreover, a large number of genes are
garding the contribution of the immune system to ische- expressed in both endothelial cells and hematopoietic
mic damages [37], the neuroprotective and healing effect precursor cells of the adult organism [49,50]. Three
of immune-cell activation [38,39], the protective role hours after occlusion, Semax altered the expression of
of the immune system, and its therapeutic function genes associated with the artery vasodilation process as
[40-42]. It cannot be ruled out that the observed effect well. Our earlier studies showed that capillary bore ex-
of Semax on brain stroke can be explained by its impact tension was observed as early as 15 min after the admin-
on protective immune mechanisms. Some recent reports istration of the peptide [9].
have described interactions between nervous tissues and As shown in Figure 3B, 24 h after occlusion, Semax
the immune system, which were observed after the ad- affected the development of the endothelial tissue and
ministration of neuropeptides. For instance, the noo- the migration of smooth muscle cells, which was an in-
tropic medication cerebrolysin favored the survival of dication of vessel formation and stabilization [48]. Fi-
immunocompetent cells [43]. Another preparation, the nally, another biological process, i.e., the activation of
vasoactive intestinal peptide (VIP), which has neuro- blood cells, was affected by Semax 24 h after pMCAO,
trophic effects, acted as an immunomodulator [44]. The which followed logically after the process of the forma-
possible effect of neuromodulation on the consequences tion of blood cells induced by Semax 3 h after the
of ischemia is believed to be real, although it has not occlusion.
been studied sufficiently [45]. Thus, as demonstrated here, the action of Semax on
the expression of genes that ensure the formation and
functioning of the vascular system in ischemic condi-
Response of the vascular system to the administration of tions also uncovered its possible vascular and regenera-
the neuropeptide Semax tive properties, in addition to its neuroprotective and
Here, we found changes in the expression levels of sev- vasoactive effects.
eral genes involved in the functioning of the vascular
system as a response to Semax administration. The for-
mation of new blood vessels in the ischemized areas rep- Conclusions
resents one of the approaches used in the treatment of In this study, we analyzed the action of the neuroprotec-
brain stroke [46]. It should be mentioned that the pres- tive peptide Semax on the transcriptome of rat brain cor-
ence of immune cells in the damaged tissues is a typical tical cells in conditions of experimental focal ischemia.
Table 4 Genes related to the vascular system and exhibited Semax-induced alteration of expression levels
Genes related to the vascular system (24 genes): Gene symbol ENTREZ GENE_ID Fold change P-value
Cysteine-rich, angiogenic inducer, 61, mRNA. Cyr61 83476 2.43 1.5E-07
Activating transcription factor 3, mRNA. Atf3 25389 2.24 3.9E-03
Kruppel-like factor 4 (gut), mRNA. Klf4 114505 2.23 2.0E-03
A disintegrin-like and metallopeptidse (reprolysin type) with Adamts1 79252 2.18 5.2E-11
thrombospondin type 1 motif, 1, mRNA.
FBJ murine osteosarcoma viral oncogene homolog, mRNA. Fos 314322 2.11 2.0E-04
Jun B proto-oncogene, mRNA. Junb 24517 2.07 1.8E-06
Nuclear factor, interleukin 3 regulated, mRNA. Nfil3 114519 2.03 5.4E-08
Prostaglandin-endoperoxide synthase 2, mRNA. Ptgs2/Cox2 29527 2.00 8.3E-34
Brain derived neurotrophic factor, mRNA. Bdnf 24225 1.88 2.0E-04
SWI/SNF related, matrix associated, actin dependent Smarca5 307766 1.74 2.9E-09
regulator of chromatin, subfamily a, member 5, mRNA.
Zinc finger protein 36, mRNA. Zfp36 79426 1.73 5.5E-03
3h Early growth response 2, mRNA. Egr2 114090 1.70 6.6E-03

Dual specificity phosphatase 1, mRNA. Dusp1 114856 1.67 3.0E-08
Heat shock 27 kDa protein 1, mRNA. Hspb1 24471 1.63 9.0E-03
Intercellular adhesion molecule 1, mRNA. Icam1 25464 1.61 9.9E-05
Secretogranin II (chromogranin C), mRNA. Scg2 24765 1.60 3.6E-04
Relaxin 1, mRNA. Rln1 25616 1.58 4.0E-03
Amyloid beta (A4) precursor protein, mRNA. App 54226 0.66 1.9E-03
Mitogen activated protein kinase 10, mRNA. Mapk10 25272 0.66 2.6E-03
Fibronectin 1, mRNA. Fn1 25661 0.63 5.1E-04
Catalase, mRNA. Cat 24248 0.51 2.9E-05
CD74 antigen (invariant polpypeptide of MHC class II Cd74 25599 0.50 1.6E-09
antigen-associated), mRNA.
Genes related to the vascular system (12 genes):
24h
Nephroblastoma overexpressed gene, mRNA. Nov 81526 1.64 3.2E-06
CD74 antigen (invariant polpypeptide of MHC class II Cd74 25599 1.63 5.3E-06
antigen-associated), mRNA.
Secreted phosphoprotein 1, mRNA. Spp1 25353 1.58 3.4E-06
Plasminogen activator, tissue, mRNA. Plat 25692 0.65 1.1E-07
Podoplanin, mRNA. Pdpn 54320 0.63 5.3E-10
A disintegrin-like and metallopeptidse (reprolysin type) Adamts1 79252 0.56 7.5E-09
with thrombospondin type 1 motif, 1, mRNA.
Although Semax has been shown to be effective in brain As shown here, Semax influenced various biological
stroke therapy, the molecular mechanisms underlying its processes that contribute to the functioning of the differ-
neuroprotective action remain unknown. ent systems of the organism. The immune response was
most markedly affected by the action of Semax. The artery (MCA) was exposed via the transtemporal approach.
peptide increased the amount and mobility of immune A 1.5 cm scalp incision was made at the midpoint between
cells and enhanced the expression of chemokine and im- the right eye and the right ear. The temporalis muscle was
munoglobulin genes. separated in the plane of its fiber bundles and retracted in
Our data showed that Semax is likely to influence pro- order to expose the zygoma and squamosal bone. Using
cesses that accompany the formation of new blood vessels microsurgical technques, a burr hole, 2 mm in diameter,
during early ischemia cascade stages, as well as their was made with a dental drill 1 mm rostal to the anterior
stabilization at later stages. junction of the zygoma and the squamosal bone. The dura
The expression of genes responsible for the intracellular mater was carefully pierced with a scalpel. The exposed
level of Ca2+ was sensitive to Semax administration MCA was isolated and occluded by short coagulation
against the background of the unfolding pMCAO-induced using a bipolar coagulator. The craniotomy was covered
neurodegenerative processes. Our results showed that with a small piece of gelfoam, the temporalis muscle and
Semax enhanced the expression of genes encoding protein overlying skin were allowed to fall back and were sutured
products that promote intracellular Ca2+ accumulation. separately. After suturing, rats were returned to their
Possibly, the neuroprotective effect of Semax on ischemia- cages until sacrifice. The operation was last about 30 min.
damaged nervous tissues includes an impact on processes
involved in the incorporation of Ca2+ into cells. Experimental groups
Thus, the immunomodulating effects of Semax de- Animals were divided into two groups: (1) “ischemia”
scribed here, as well as its influence on the vascular sys- and (2) “ischemia + Semax” groups. pMCAO was per-
tem in conditions of ischemia, are likely to be key formed in all animals. During the experiment, ischemia
factors in the neuroprotective effects of the peptide. It + Semax animals were given intraperitoneal injections of
cannot be ruled out that the large amount of genes that Semax (100 μg/kg), whereas ischemia animals were
exhibited changed levels of expression, the functions of injected with saline. The injections of Semax or saline
which remain unknown or not well studied, will help were performed 15 min, 1, 4 and 8 h after pMCAO.
disclose other, hitherto unknown pathways of Semax ac- The rats were decapitated under anesthesia with ethyl
tion on damaged brain tissues. We must state at the ether 3 and 24 h after the operation. According to data
same time that the baffling complexity of the multicom- from the literature, significant events in the formation of
ponent nature of cerebral ischemia and the ability of a stroke area, such as excitotoxicity, mitochondrial dam-
Semax to affect a large number of biological processes age, emergence of reactive oxygen species, and apop-
require future research to uncover the full scope of the tosis, occur within the first 3 h after occlusion of an
mechanisms of action of this peptide. artery [53], and the expression of genes at the early stage
of ischemia can be studied at this time point. At the
Methods 24 h time point the infarction area reaches its maximal
Animals dimensions and the formation of the penumbra is com-
All experimental protocol were approved by Bioethics pleted [54].
Comission of Lomonosov Moscow State University in Each time point included at least five animals. We iso-
accordance with the National Institutes of Health Guide lated the frontoparietal cortex of the ischemic animals,
for the Care and Use of Laboratory Animals (NIH Publ. in which, according to histological analysis of our earlier
no. 80–23, revised 1996). We used adult male Wistar research, the damaged area was localized [51]. Total
rats (270–320 g) maintained on a 12 h light/dark cycle RNA was isolated from tissue samples.
at a temperature of 22–24°C with free access to food
and water. Microarray data analysis
Microarray experiments were carried out at ZAO
Focal cerebral ischemia model ''Genoanalytica'', Moscow, Russia. Total RNA was isolated
We applied the model of “focal cerebral ischemia” in- from tissue samples using guanidine thiocyanate [55].
duced as previously described [51]. The irreversible elec- RNA integrity was assessed by comparison with the rRNA
trical coagulation of the distal segment of the left middle bands obtained in agarose gel electrophoresis under de-
cerebral artery was performed under anesthesia with naturing conditions. RNA was quantified using Nano-
chloral hydrate (300 mg/kg). Drop, and its quality was assessed using an Agilent RNA
Focal cerebral ischemia was induced by direct pMCAO, 6000 Nano Chip. Total RNA (400 ng) was amplified using
involving craniotomy technique as previously described an Illumina® TotalPrep™ RNA Amplification Kit (Ambion,
[52] without occlusion of carotid artery. In detail, anesthesia USA) containing 22,523 probes for a total of 22,228 rat
was induced by intraperitoneal administration of chloral genes selected primarily from the NCBI Reference Se-
hydrate (400 mg/kg body weight). The left middle cerebral quence database (Illumina, USA). The Illumina RatRef-12
Expression BeadChip was used in accordance with the revising manuscript critically for content; SL - have given final approval of
manufacturer’s instructions. The BeadArray Reader was the version to be published. All authors read and approved the final
manuscript.
employed for data acquisition, and the analysis was ac-
complished with the help of the Genome Studio software
Acknowledgements
(Illumina, USA) using the gene-expression module. The This study was partially supported by grants of the Russian Foundation for Basic
statistical algorithm used in GenomeStudio gene expres- Research (11-04-00843, 12-04-31528, 13-04-40083-Н), and the “Molecular and
sion analysis is the Illumina Custom error model. Cell Biology” Program of the Russian Academy of Sciences, and the Federal
Program for Support of Scientific Schools of the Russian Ministry of Science and
Education.
Functional analysis
The interactive Web-based Ingenuity iReport program Author details
1
Human Molecular Genetics Department, Institute of Molecular Genetics,
[12] based on Fisher’s exact test (P-value < 0.01) was ap- Russian Academy of Sciences, Moscow, Russian Federation. 2Institute of
plied to identify the molecular functions of the products Cerebrovascular Pathology and Stroke, Pirogov Russian National Research
of the genes that exhibited altered expression in the con- Medical University, Moscow, Russian Federation. 3Faculty of Medicine,
Moscow State University n/a M.V. Lomonosov, Moscow, Russian Federation.
ditions established, as well as signaling pathways and
statistically significant biological processes. Ingenuity Received: 14 June 2013 Accepted: 18 March 2014
iReport helps the quick identification of especially sig- Published: 24 March 2014
nificant genes, signaling pathways, and processes that
are most relevant to the experimental data. Only those References
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The Peptide Semax Affect The Expression Genes Related To The Immune and Vascular System

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The peptide semax affects the expression of

3h Early growth response 2, mRNA. Egr2 114090 1.70 6.6E-03

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